This invention relates generally to genetic engineering involving recombinant DNA technology, and particularly to the identification of nucleic acid molecules capable of hybridizing under stringent conditions to the nucleotide sequence of the hBub1 cDNAs shown in SEQ ID NO: 1 or GenBank Accession NO: AF043294.
Eukaryotic cells respond to internal demands or sense the external environment by remaining quiescent, staying in the cell cycle, or activating cell death programs. The coordination of such a complex process necessarily entails very sensitive and sophisticated regulation. Increasing evidence indicates that protein kinases and phosphatases are central to the regulation of these events (1-3).
Extensive research in the past has led to the identification of cellular mechanisms (commonly referred to as checkpoints) that monitor the readiness of a cell to enter the next stage of the cell cycle (4,5). There are two major checkpoints controlling the G2M transition and M-phase progression. In yeast, cdc2/CDC28 kinase is pivotal in the G2/M transition (3) while the spindle checkpoint kinase BUB1 is thought to be involved in regulating mitotic progression (6,7). Genetic analyses have identified six distinct yeast genes (BUB1, 2, and 3 and MAD1, 2, and 3) that are important in regulating the spindle checkpoint (6-8). BUB1 encodes a protein serine/threonine kinase and is itself phosphorylated when the cell enters mitosis (9). A recent study shows that Bub1p activates the spindle checkpoint in the budding yeast (10). BUB2 is structurally related to the fission yeast cdc16 gene product, which plays an essential part in cytokinesis (11). BUB3, unrelated to any other known proteins, appears to be a substrate of BUB1 (9). MAD1 protein is hyperphosphorylated when wild type yeast cells are arrested in mitosis upon disruption of microtubules (12). MAD2 is required for MAD1 hyperphosporylation in yeast (7,12), and yeast MAD3 is a 60-kDa protein whose biochemical function remains unknown (7).
Until recently, little was known regarding the molecular components in spindle checkpoint regulation in high eukaryotes. It has been shown that human Mad2, structurally and functionally conserved (13), is localized at the kinetochore after chromosome condensation but not after metaphase (13). XMAD2 from Xenopus is co-localized with unattached kinetochores in prometaphase but disappeared from the apparatus in metaphase (14). It has also been demonstrated that human Mad2 expression is down-regulated in a human breast cancer cell line that is sensitive to taxol and nocodazole (13). Murine Bub1 (mnBub1) has recently been cloned and characterized (15). The mBub1 protein also localizes to the kinetochore during early mitosis (15). When mBub1 function is compromised, cells are unable to appropriately initiate programmed cell death when an apoptotic signal is present (15). The hBub1 gene has recently been implicated in the development of certain colorectal cancers (16).
The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human gene (hBub1), some alleles of which cause susceptibility to cancer. More specifically, the present invention relates to germline mutations in the hBub1 gene and their use in the diagnosis of predisposition to cancer. The invention further relates to somatic mutations in the hBub1 gene in human cancer and their use in the diagnosis and prognosis of human cancer. Additionally, the invention relates to somatic mutations in the hBub1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the hBub1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the hBub1 gene for mutations, which are useful for diagnosing the predisposition to cancer.
The present invention provides an isolated polynucleotide comprising all, or a portion of the hBub1 locus or of a mutated hBub1 locus, preferably at least eight bases and not more than about 100 kb in length. Such polynucleotides may be antisense polynucleotides. The present invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell.
Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the hBub1 locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the hBub1 locus, and may further include a step of providing a set of polynucleotides that are primers for amplification of said portion of the hBub1 locus. The method is useful for either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.
The present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the hBub1 locus.
The present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the hBub1 locus, the kits comprising a polynucleotide complementary to the portion of the hBub1 locus packaged in a suitable container, and instructions for its use.
The present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the hBub1 locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the hBub1 locus.
The present invention further provides methods of screening the hBub1 gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the hBub1 locus, and may further include a step of providing a set of polynucleotides that are primers for amplification of said portion of the hBub1 locus. The method is useful for identifying mutations for use in either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.
The present invention further provides methods of screening suspected hBub1 mutant alleles to identify mutations in the hBub1 gene.
In addition, the present invention provides methods of screening drugs for cancer therapy to identify suitable drugs for restoring hBub1 gene product function.
Finally, the present invention provides the means necessary for production of gene-based therapies directed at cancer cells. These therapeutic agents may take the form of polynucleotides comprising all or a portion of the hBub1 locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the hBub1 protein is reconstituted. Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of hBub1. These may functionally replace the activity of hBub1 in vivo.